Regulation of gene expression by SnRK1 kinases and miRNAs during the plant stress response

Dissertation presented to obtain the Ph.D degree in Plant Physiology

A-kinase anchoring proteins: Molecular regulators of the cardiac stress response

In response to stress or injury the heart undergoes a pathological remodeling process, associated with hypertrophy, cardiomyocyte death and fibrosis, that ultimately causes cardiac dysfunction and heart failure. It has become increasingly clear that signaling events associated with these pathological cardiac remodeling events are regulated by scaffolding and anchoring proteins, which allow coordination of pathological signals in space and time. A-kinase anchoring proteins (AKAPs) constitute a family of functionally related proteins that organize multiprotein signaling complexes that tether the cAMP-dependent protein kinase (PKA) as well as other signaling enzymes to ensure integration and processing of multiple signaling pathways. This review will discuss the role of AKAPs in the cardiac response to stress. Particular emphasis will be given to the adaptative process associated with cardiac hypoxia as well as the remodeling events linked to cardiac hypertrophy and heart failure. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.

An inhibitor of HIV-1 protease modulates constitutive eIF2α dephosphorylation to trigger a specific integrated stress response.

Inhibitors of the HIV aspartyl protease [HIV protease inhibitors (HIV-PIs)] are the cornerstone of treatment for HIV. Beyond their well-defined antiretroviral activity, these drugs have additional effects that modulate cell viability and homeostasis. However, little is known about the virus-independent pathways engaged by these molecules. Here we show that the HIV-PI Nelfinavir decreases translation rates and promotes a transcriptional program characteristic of the integrated stress response (ISR). Mice treated with Nelfinavir display hallmarks of this stress response in the liver, including α subunit of translation initiation factor 2 (eIF2α) phosphorylation, activating transcription factor-4 (ATF4) induction, and increased expression of known downstream targets. Mechanistically, Nelfinavir-mediated ISR bypassed direct activation of the eIF2α stress kinases and instead relied on the inhibition of the constitutive eIF2α dephosphorylation and down-regulation of the phophatase cofactor CReP (Constitutive Repressor of eIF2α Phosphorylation; also known as PPP1R15B). These findings demonstrate that the modulation of eIF2α-specific phosphatase cofactor activity can be a rheostat of cellular homeostasis that initiates a functional ISR and suggest that the HIV-PIs could be repositioned as therapeutics in human diseases to modulate translation rates and stress responses.

The role of the peptidyl prolyl isomerase Rrd1 in the transcriptional stress response

La régulation de la transcription est un processus complexe qui a évolué pendant des millions d’années permettant ainsi aux cellules de s’adapter aux changements environnementaux. Notre laboratoire étudie le rôle de la rapamycine, un agent immunosuppresseur et anticancéreux, qui mime la carence nutritionelle. Afin de comprendre les mécanismes impliqués dans la réponse a la rapamycine, nous recherchons des mutants de la levure Saccaromyces cerevisiae qui ont un phenotype altérée envers cette drogue. Nous avons identifié le gène RRD1, qui encode une peptidyl prolyl isomérase et dont la mutation rend les levures très résistantes à la rapamycine et il semble que se soit associé à une réponse transcriptionelle alterée. Mon projet de recherche de doctorat est d’identifier le rôle de Rrd1 dans la réponse à la rapamycine. Tout d’abord nous avons trouvé que Rrd1 interagit avec l’ARN polymérase II (RNAPII), plus spécifiquement avec son domaine C-terminal. En réponse à la rapamycine, Rrd1 induit un changement dans la conformation du domaine C-terminal in vivo permettant la régulation de l’association de RNAPII avec certains gènes. Des analyses in vitro ont également montré que cette action est directe et probablement liée à l’activité isomérase de Rrd1 suggérant un rôle pour Rrd1 dans la régulation de la transcription. Nous avons utilisé la technologie de ChIP sur micropuce pour localiser Rrd1 sur la majorité des gènes transcrits par RNAPII et montre que Rrd1 agit en tant que facteur d’élongation de RNAPII. Pour finir, des résultats suggèrent que Rrd1 n’est pas seulement impliqué dans la réponse à la rapamycine mais aussi à differents stress environnementaux, nous permettant ainsi d’établir que Rrd1 est un facteur d’élongation de la transcription requis pour la régulation de la transcription via RNAPII en réponse au stress.

Heat stress response for physiological traits in dairy and dual purpose cattle populations on phenotypic and genetic scales

The main objective of this thesis was to determine the potential impact of heat stress (HS) on physiological traits of lactating cows and semen quality of bulls kept in a temperate climate. The thesis is comprised of three studies. An innovative statistical modeling aspect common to all three studies was the application of random regression methodology (RRM) to study the phenotypic and genetic trajectory of traits in dependency of a continuous temperature humidity index (THI). In the first study, semen quality and quantity traits of 562 Holstein sires kept on an AI station in northwestern Germany were analyzed in the course of THI calculated from data obtained from the nearest weather station. Heat stress was identified based on a decline in semen quality and quantity parameters. The identified general HS threshold (THI = 60) and the thermoneutal zone (THI in the range from 50 to 60) for semen production were lower than detected in studies conducted in tropical and subtropical climates. Even though adult bulls were characterized by higher semen productivity compared to younger bulls, they responded with a stronger semen production loss during harsh environments. Heritabilities (low to moderate range) and additive genetic variances of semen characteristics varied with different levels of THI. Also, based on genetic correlations genotype, by environment interactions were detected. Taken together, these findings suggest the application of specific selection strategies for specific climate conditions. In the second study, the effect of the continuous environmental descriptor THI as measured inside the barns on rectal temperatures (RT), skin temperatures (ST), vaginal temperatures (VT), respiration rates (RR), and pulse rate (PR) of lactating Holstein Friesian (HF) and dual-purpose German black pied cattle (DSN) was analyzed. Increasing HS from THI 65 (threshold) to THI 86 (maximal THI) resulted in an increase of RT by 0.6 °C (DSN) and 1 °C (HF), ST by 3.5 °C (HF) and 8 °C (DSN), VT by 0.3 °C (DSN), and RR by 47 breaths / minute (DSN), and decreased PR by 7 beats / minute (DSN). The undesired effects of rising THI on physiological traits were most pronounced for cows with high levels of milk yield and milk constituents, cows in early days in milk and later parities, and during summer seasons in the year 2014. In the third study of this dissertation, the genetic components of the cow’s physiological responses to HS were investigated. Heat stress was deduced from indoor THI measurements, and physiological traits were recorded on native DSN cows and their genetically upgraded crosses with Holstein Friesian sires in two experimental herds from pasture-based production systems reflecting a harsh environment of the northern part of Germany. Although heritabilities were in a low range (from 0.018 to 0.072), alterations of heritabilities, repeatabilities, and genetic components in the course of THI justify the implementation of genetic evaluations including heat stress components. However, low repeatabilities indicate the necessity of using repeated records for measuring physiological traits in German cattle. Moderate EBV correlations between different trait combinations indicate the potential of selection for one trait to simultaneously improve the other physiological attributes. In conclusion, bulls of AI centers and lactating cows suffer from HS during more extreme weather conditions also in the temperate climate of Northern Germany. Monitoring physiological traits during warm and humid conditions could provide precious information for detection of appropriate times for implementation of cooling systems and changes in feeding and management strategies. Subsequently, the inclusion of these physiological traits with THI specific breeding values into overall breeding goals could contribute to improving cattle adaptability by selecting the optimal animal for extreme hot and humid conditions. Furthermore, the recording of meteorological data in close distance to the cow and visualizing the surface body temperature by infrared thermography techniques might be helpful for recognizing heat tolerance and adaptability in cattle.

Dual-specificity phosphatases in the hypo-osmotic stress response of keratin-defective epithelial cell lines

Although mutations in intermediate filament proteins cause many human disorders, the detailed pathogenic mechanisms and the way these mutations affect cell metabolism are unclear. In this study, selected keratin mutations were analysed for their effect on the epidermal stress response. Expression profiles of two keratin-mutant cell lines from epidermolysis bullosa simplex patients (one severe and one mild) were compared to a control keratinocyte line before and after challenge with hypo-osmotic shock, a common physiological stress that transiently distorts cell shape. Fewer changes in gene expression were found in cells with the severely disruptive mutation (55 genes altered) than with the mild mutation (174 genes) or the wild type cells (261 genes) possibly due to stress response pre-activation in these cells. We identified 16 immediate-early genes contributing to a general cell response to hypo-osmotic shock, and 20 genes with an altered expression pattern in the mutant keratin lines only. A number of dual-specificity phosphatases (MKP-1, MKP-2, MKP-3, MKP-5 and hVH3) are differentially regulated in these cells, and their downstream targets p-ERK and p-p38 are significantly up-regulated in the mutant keratin lines. Our findings strengthen the case for the expression of mutant keratin proteins inducing physiological stress, and this intrinsic stress may affect the cell responses to secondary stresses in patients' skin.

Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis

We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine (DCVC) resulted in a >1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.

Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis

We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine ( DCVC) resulted in a > 1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.

The role played by the interaction between genetic factors and attachment in the stress response in infancy

Background: The importance of understanding which environmental and biological factors are involved in determining individual differences in physiological response to stress is widely recognized, given the impact that stress has on physical and mental health. Methods: The child-mother attachment relationship and some genetic polymorphisms (5-HTTLPR, COMT and GABRA6) were tested as predictors of salivary cortisol and alpha amylase concentrations, two biomarkers of hypothalamic-pituitary-adrenocortical (HPA) axis and sympathetic adrenomedullary (SAM) system activity, during the Strange Situation (SS) procedure in a sample of more than 100 healthy infants, aged 12 to 18 months. Results: Individual differences in alpha amylase response to separation were predicted by security of attachment in interaction with 5-HTTLPR and GABRA6 genetic polymorphisms, whereas alpha amylase basal levels were predicted by COMT x attachment interaction. No significant effect of attachment, genetics and their interaction on cortisol activity emerged. Conclusions: These results help to disentangle the role played by both genetic and environmental factors in determining individual differences in stress response in infancy. The results also shed light on the suggestion that HPA and SAM systems are likely to have different characteristic responses to stress.

Iron homeostasis and management of oxidative stress response in bacteria

Iron is both an essential nutrient for the growth of microorganisms, as well as a dangerous metal due to its capacity to generate reactive oxygen species (ROS) via the Fenton reaction. For these reasons, bacteria must tightly control the uptake and storage of iron in a manner that restricts the build-up of ROS. Therefore, it is not surprising to find that the control of iron homeostasis and responses to oxidative stress are coordinated. The mechanisms concerned with these processes, and the interactions involved, are the subject of this review.

Intake, water consumption, ruminal fermentation, and stress response of beef heifers fed after different lengths of delays in the daily feed delivery time

Four rumen-fistulated Holstein heifers (134 +/- 1 kg initial BW) were used in a 4 x 4 Latin square design to determine the effects of delaying daily feed delivery time on intake, ruminal fermentation, behavior, and stress response. Each 3-wk experimental period was preceded by 1 wk in which all animals were fed at 0800 h. Feed bunks were cleaned at 0745 h and feed offered at 0800 h (T0, no delay), 0900 (T1), 1000 (T2), and 1100 (T3) from d1 to 21 with measurements taken during wk 1 and 3. Heifers were able to see each other at all times. Concentrate and barley straw were offered in separate compartments of the feed bunks, once daily and for ad libitum intake. Ruminal pH and saliva cortisol concentrations were measured at 0, 4, 8, and 12 h postfeeding on d 3 and 17 of each experimental period. Fecal glucocorticoid metabolites were measured on d 17. Increasing length of delay in daily feed delivery time resulted in a quadratic response in concentrate DMI (low in T1 and T2; P = 0.002), whereas straw DMI was greatest in T1 and T3 (cubic P = 0.03). Treatments affected the distribution of DMI within the day with a linear decrease observed between 0800 and 1200 h but a linear increase during nighttimes (2000 to 0800 h), whereas T1 and T2 had reduced DMI between 1200 and 1600 h (quadratic P = 0.04). Water consumption (L/d) was not affected but decreased linearly when expressed as liters per kilogram of DMI (P = 0.01). Meal length was greatest and eating rate slowest in T1 and T2 (quadratic P <= 0.001). Size of the first meal after feed delivery was reduced in T1 on d 1 (cubic P = 0.05) and decreased linearly on d 2 (P = 0.01) after change. Concentrate eating and drinking time (shortest in T1) and straw eating time (longest in T1) followed a cubic trend (P = 0.02). Time spent lying down was shortest and ruminating in standing position longest in T1 and T2. Delay of feeding time resulted in greater daily maximum salivary cortisol concentration (quadratic P = 0.04), which was greatest at 0 h in T1 and at 12 h after feeding in T2 (P < 0.05). Daily mean fecal glucocorticoid metabolites were greatest in T1 and T3 (cubic P = 0.04). Ruminal pH showed a treatment effect at wk 1 because of increased values in T1 and T3 (cubic P = 0.01). Delaying feed delivery time was not detrimental for rumen function because a stress response was triggered, which led to reduced concentrate intake, eating rate, and size of first meal, and increased straw intake. Increased salivary cortisol suggests that animal welfare is compromised.

Effect of Mussel`s Gender and Size on a Stress Response Biomarker

In mussels, stress signals such as heat, osmotic shock and hypoxia lead to the activation of the phosphorylated p38 mitogen activated protein kinase (pp38-MAPK). This stress activated protein has been efficiently used as a biomarker to several natural and anthropogenic stresses. However, what has not been tested is whether differences in gender or size can affect the response of this biomarker. The present study tested whether there was variation in the expression of pp38-MAPK in mussels Perna perna of different gender and size classes when exposed to natural stress conditions, such as air exposure. The results show that gender does not affect the expression of pp38-MAPK. However, size does have an effect, where mussels smaller than 6.5 cm displayed significantly (p < 0.05) lower levels of pp38-MAPK when compared to those larger than 7 cm. Mussels are one of the most used bioindicator species and the use of biomarkers to determine the health status of an ecosystem has been greatly increasing over the years. The present study highlights the importance of using mussels of similar size classes when performing experiments using stress-related biomarkers.

A two-component system, an anti-sigma factor and two paralogous ECF sigma factors are involved in the control of general stress response in Caulobacter crescentus

The extracytoplasmic function sigma factor sigma(T) is the master regulator of general stress response in Caulobacter crescentus and controls the expression of its paralogue sigma(U). In this work we showed that PhyR and NepR act, respectively, as positive and negative regulators of sigma(T) expression and function. Biochemical data also demonstrated that NepR directly binds sigma(T) and the phosphorylated form of PhyR. We also described the essential role of the histidine kinase gene CC3474, here denominated phyK, for expression of sigma(T)-dependent genes and for resistance to stress conditions. Additionally, in vivo evidence of PhyK-dependent phosphorylation of PhyR is presented. This study also identified a conserved cysteine residue (C95) located in the periplasmic portion of PhyK that is crucial for the function of the protein. Furthermore, we showed that PhyK, PhyR and sigma(T) regulate the same set of genes and that sigma(T) apparently directly controls most of its regulon. In contrast, sigma(U) seems to have a very modest contribution to the expression of a subset of sigma(T)-dependent genes. In conclusion, this report describes the molecular mechanism involved in the control of general stress response in C. crescentus.

Trypanosoma cruzi MSH2: Functional analyses on different parasite strains provide evidences for a role on the oxidative stress response

Components of the DNA mismatch repair (MMR) pathway are major players in processes known to generate genetic diversity, such as mutagenesis and DNA recombination. Trypanosoma cruzi, the protozoan parasite that causes Chagas disease has a highly heterogeneous population, composed of a pool of strains with distinct characteristics. Studies with a number of molecular markers identified up to six groups in the T. cruzi population, which showed distinct levels of genetic variability. To investigate the molecular basis for such differences, we analyzed the T. cruzi MSH2 gene, which encodes a key component of MMR, and showed the existence of distinct isoforms of this protein. Here we compared cell survival rates after exposure to genotoxic agents and levels of oxidative stress-induced DNA in different parasite strains. Analyses of msh2 mutants in both T. cruzi and T. brucei were also used to investigate the role of Tcmsh2 in the response to various DNA damaging agents. The results suggest that the distinct MSH2 isoforms have differences in their activity. More importantly, they also indicate that, in addition to its role in MMR, TcMSH2 acts in the parasite response to oxidative stress through a novel mitochondrial function that may be conserved in T. brucei. (C) 2010 Elsevier B.V. All rights reserved.